In the field of biological sciences, amplification of DNA or RNA has been carried out for various purposes. For example, competitive PCR (A. Wang, et al., Proc. Natl. Acad. Sci. USA, 86, 9717-9721, 1989) and real-time PCR (S. H. Aliyu, et al., Journal of Antimicrobial, 54, 968, 2004) have been known as techniques for analyzing the expression of given genes and quantifying the expression levels thereof. These techniques involve the application of a common nucleic acid amplification technique, i.e., polymerase chain reaction (PCR) (R. K. Saiki, et al., Science, 239, 487-491, 1988), to determine expression levels based on the amplified genes.
The aforementioned nucleic acid amplification technique for analysis consists of 3 steps of denaturation from double-strand template DNA to single-strand template DNA, annealing of the primers to the single-strand template DNA, and elongation of complimentary strands from the primers. It can also consist of 2 steps of denaturation and elongation. Such amplification technique requires repetition of a cycle from high temperature treatment to low temperature treatment.
In order to carry out such a cycle, it is necessary to carry out PCR with the use of a thermal cycler that is capable of accurate temperature control. An increased number of cycles results in a prolonged time frame required for bringing the temperature of the apparatus and that of the reaction solution to the determined levels. This disadvantageously prolongs the time frame required for analysis.
In order to overcome such drawbacks, nucleic acid amplification techniques that could be carried out under isothermal conditions were developed. Examples of major techniques that have been known include nucleic acid sequence-based amplification (NASBA) (J. Compton, et al., Nature, 350, 91-92, 1991), strand displacement amplification (SDA) (G. T. Walker, et. al., Proc. Natl. Acad. Sci USA, 89, 392-396, 1992), self-sustained sequence replication (3SR) (J. C. Guatelli, et al., Proc Natl. Acad. Sci. USA, 87, 1874-1878, 1990), transcription-mediated amplification (TMA) (JP Patent No. 3241717), and Qβ replicase amplification (JP Patent No. 2710159). In these isothermal nucleic acid amplification techniques, primer elongation and primer annealing to a single-strand elongation product are carried out in a reaction mixture that is maintained at a constant temperature.
Among these techniques, TMA, Qβ replicase amplification, 3SR, and NASBA techniques, whereby RNA is amplified in the end, involve the use of RNA polymerase or reverse transcriptase to amplify the target nucleic acid sequence in a sample. Since these techniques do not comprise a high temperature cycle for accelerating denaturation during the reaction, a template has a secondary structure and thus annealing of a primer to the template may not be satisfactorily carried out. Thus, these techniques cannot always produce an amplified product even with the use of a primer that can yield an amplified product of interest by PCR. For the same reason, it is difficult to detect an amplified product of a sequence to be detected. Even though primer and probe sequences were designed at regions of interest, therefore, amplification or detection efficiency is not always satisfactory, and designing of the primers or probes that could be employed for isothermal amplification was difficult. As is apparent from the foregoing explanation, conventional isothermal amplification techniques have various drawbacks such as the difficulty of designing primers and probes that could effectively produce and detect amplified products with high sensitivity. Therefore, development of a technique of isothermal nucleic acid amplification has been awaited in order to overcome such drawbacks.